1. Field of the Invention
This invention relates to the quantitative determination of substances in or characteristics of liquid media, including body fluids such as serum, based on specific binding assay techniques. In particular, the invention is directed to the detection of antigens or haptens based on immunoassay techniques involving the use of labeled reagents, such as radiolabeled reagents. The present invention provides an improved method of performing the separation of bound- and free-label inherent in heterogeneous specific binding assays.
2. Description of the Prior Art
A living system is able to detect, recognize and respond to the presence of foreign material (antigen) such as protein, virus, bacteria, and so forth, within that system. This response takes, inter alia, the form of producing an antibody specific for the particular antigen. There then occurs a specific reaction between the antibody and the antigen to form a complex. An antibody once produced is also capable of binding a hapten, i.e., a relatively small and simple compound which may be the determinant group of a given antigen, which hapten is capable of binding with the specific antibody but incapable itself of giving rise to the production of an antibody, unless it is bound to an antigenic carrier.
The binding interaction between an antigen or a hapten and its antibody is specific and sensitive. Other types of materials that participate in similar specific and sensitive binding interactions are enzymes and their substrates; materials such as hormones, vitamins, metabolites and pharmacological agents, and their receptors and binding substances; and other substances known in the science. These specific and sensitive binding reactions have given rise to a rapidly emerging analytical technique known as the specific binding assay technique. In one such type of assay method, the substance, or group of substances, to be determined (herein referred to as "ligand") in a liquid sample is placed in competition with a labeled form of the ligand or of a binding analog thereof for binding to a binding reagent. Where a radioactive label is used and the binding reagent is an antibody, the method is known as a radioimmunoassay method. Recently, several alternative labeling materials have been reported for replacement of radioisotopes, including enzymes, coenzymes, enzyme substrates, enzyme modulators such as inhibitors and allosteric effectors, fluorescent molecules, luminescent molecules, and others. For illustrative purposes, the discussion which follows describes one particular type of specific binding assay technique, a competitive binding radioimmunoassay technique.
This system consists of antigen or hapten labeled with a radioactive marker, unlabeled native antigen (in the test sample) and specific antibody whereby there is competition between the unlabeled antigen and the labeled antigen for binding to a limited amount of antibody. Hence, the greater the concentration of unlabeled antigen from the test sample in the system, the less the labeled antigen will be bound by the antibody. This may be diagrammatically represented as follows: ##STR1##
If the concentration of labeled antigen and antibody is fixed and the only variable is the level of unlabeled antigen, it becomes possible to establish an assay system for measuring the unknown level of unlabeled antigen by physically separating the antigen-antibody complex from the remaining free antigen (both labeled and unlabeled). The radioactivity of the unknowns is compared with a standard curve plotting of the values given by a range of known amounts of the antigen treated in the same manner.
There are many known procedures for separating the free unbound antigen or hapten from the complex antigen-antibody. One method known as chromatoelectrophoresis combines techniques of paper chromatography and paper electrophoresis. Paper with a high affinity for the free antigen (such as Whatman 3 MM, Whatman 3 MC and DEAE paper) are used as carriers. While this technique is discriminative and has been used in the assay of insulin, growth hormone, glucagon, parathyroid hormone, thyroid stimulating hormone and other peptide hormones, it has a number of prominent disadvantages which limits its use. A limited amount of material may be applied to the adsorbent, and the separation is both laborious and time-consuming.
Another known procedure, which has been proposed for the assay of the above-mentioned peptide hormones, makes use of ascending paper-wick chromatography, e.g., on Whatman 3MC and DE-cellulose paper or on paper loaded with weak ion-exchange resins. Orskov, Scand. J. Clin. Lab. Invest. 20: 297(1967). This method too has the disadvantage that the amount of sample which can be applied to the lower tip of the paperwick is comparatively small. It is further necessary in this technique to dry the paper (sometimes even twice) and to cut it before the counting, which is disadvantageous for applying the method to the assay of a large number of samples by mechanical means. It is also most significant that in more than a decade this known method has not been cited as being of any importance in the literature and has not been applied to the radioimmunoassay of relatively low-molecular weight haptens.
By another known method the antigen-antibody complex is precipitated by salts, organic material or solvents under conditions that do not affect the free antigen. Among the salts, materials and solvents used are: ethanol, acetone, sodium sulfate, ammonium sulfate, dioxane, trichloroacetic acid, polyethylene glycol, and so forth. The use of salts, solvents or organic materials has the advantage that the separation is immediate, and a second incubation is not necessary. However, the chemical precipitation technique may cause the co-precipitation of other proteins, often causing an incomplete separation of the two fractions.
There is also known the double antibody technique, which is widely used for the separation of the bound and the free antigen. By this method a second antibody that was raised against the first antibody is used to precipitate the primary antigen-antibody complex. For example, if the first antibody was raised in a rabbit then the second antibody may be an antiserium to rabbit gammaglobulin raised in goats. One disadvantage of this technique is that the use of a second antibody introduces an additional incubation. Specific binding assay methods employing a double antibody separation technique are described in U.S. Pat. Nos. 3,839,153 and 3,872,225.
Furthermore, there are known various solid-phase techniques for the separation of free and bound antigen. These techniques make use of antibodies covalently bound or physically adsorbed to an insoluble matrix (immunosorbents), such as bentonite, cellulose, bromacetyl cellulose, the crosslinked dextrans (Sephadex), sepharose, plastic (non-cross-linked polystyrene or polypropylene) beads, Enzacryl AA, nitro-cellulose membranes, and so forth. The formed antibody-antigen complex is held by the solid phase and the bound fraction is thus directly separated from the free fraction.
By yet another method the free (unbound) antigens are bound to adsorbents which then can be precipitated by centrifugation. Powdered talc (magnesium silicate), Kaolin (aluminum silicate), QUSO (microgranules of silica), cellulose powder, and so forth, are some of the simple adsorbents used. Many separations are performed by using adsorbent charcoal coated with dextran. The dextran behaves rather like a sieve which allows the smaller molecules of free antigen to pass and these are then bound by the charcoal, leaving the bound antigen in solution, after the charcoal has been removed by centrifugation or filtration.
It is also known to use ion exchange or other types of resins to bind free antigens by electrostatic forces and this method has been used so far mainly for the determination of small molecules such as thyroid hormones (T-3 and T-4). Examples of this type of methodology are described in U.S. Pat. Nos. 3,659,104; 3,710,117 and 3,961,894.
One technique of this type used for the separation of the antigen-antibody complex from free antigen employs a column packed with material which preferentially adsorbs either the free antigen or the antigen-antibody complex. The incubated aqueous reaction mixture is applied to the head of such a column and the column is then eluted. The radioactivity of either the column or the eluate is then determined and the content of the antigen in the starting solution is calculated from the count.
In practice it has been found that this technique is somewhat cumbersome and not well suited for the rapid performance of a large number of radioimmunoassays with the aid of mechanical means. One of the reasons for this is that it is necessary to wash the non-adsorbed component completely out of the column which takes time and requires a relatively large amount of buffer solution.
It is an object of the present invention to provide an improved specific binding assay method in which the separation of the bound-species of the labeled component and the free-species thereof is accomplished in a novel manner which is more advantageous than the separation methods known in the art.